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Targeting the MUC1-C Oncoprotein Downregulates HER2 Activation and Abrogates Trastuzumab Resistance in Breast Cancer Cells

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Targeting the MUC1-C Oncoprotein Downregulates HER2 Activation and Abrogates Trastuzumab Resistance in Breast Cancer Cells Oncogene (2014) 33, 3422–3431 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells D Raina1, Y Uchida2, A Kharbanda2, H Rajabi2, G Panchamoorthy1, C Jin2, S Kharbanda1, M Scaltriti3, J Baselga3 and D Kufe2 Patients with HER2-positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane mucin 1 (MUC1) oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1 C-terminal subunit (MUC1-C) in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in the downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1–C/HER2 complexes and decreases in tyrosine-phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab- resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (B20-fold) as compared with that in sensitive cells. In addition, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with the downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance. Oncogene (2014) 33, 3422–3431; doi:10.1038/onc.2013.308; published online 5 August 2013 Keywords: MUC1; HER2; HER3; trastuzumab; resistance; breast cancer INTRODUCTION that resistance of HER2-overexpressing breast cancer cells to The HER2/ERBB2 receptor tyrosine kinase (RTK) is overexpressed in trastuzumab is conferred by (i) upregulation of cyclin E and an approximately 20% of human breast cancers and is associated increase in CDK2 activity,20 and (ii) decreased expression of the with aggressive disease and poor survival.1,2 HER2 forms PPM1H phosphatase that regulates stability of the CDK inhibitor heterodimers with HER3 and thereby phosphorylates and p27.21,22 These findings have provided the experimental basis for activates HER3.3,4 Trastuzumab is a humanized monoclonal designing strategies that target pathways associated with antibody that binds to the HER2 extracellular domain and trastuzumab resistance to reverse unresponsiveness to this agent. destabilizes ligand-independent HER2/HER3 complexes.5 Mucin 1 (MUC1) is a heterodimeric protein that associates with Targeting of HER2 with trastuzumab in HER2-overexpressing HER2 at the surface of breast cancer cells.23,24 MUC1 is translated breast cancer cells also decreases HER2 levels6,7 and induces G1 as a single polypeptide that undergoes autocleavage into arrest by stabilizing the CDK inhibitor p27.8 Trastuzumab extends N-terminal (MUC1-N) and C-terminal (MUC1-C) fragments, which the overall survival of certain patients with HER2-overexpressing in turn form a stable complex at the cell membrane.24,25 The breast cancers when used as monotherapy or in combination with MUC1-N/MUC1-C heterodimer is positioned at the apical border of chemotherapy.9–11 However, many patients exhibit de novo breast epithelial cells and is sequestered from RTKs that are unresponsiveness to trastuzumab or develop acquired resistance expressed at the basolateral membranes.24,25 However, with loss after treatment.11 Trastuzumab resistance has been associated of apical–basal polarity as a result of stress or transformation, with constitutive activation of the phosphatidylinositol-3 kinase MUC1 is repositioned over the entire cell membrane and interacts pathway as a result of phosphatase and tensin homolog (PTEN) with RTKs such as HER2.23–25 MUC1-N, the mucin component of deficiency12 or PIK3CA gene mutations.13 Phosphatase and tensin the heterodimer, is shed from the cell surface.24,25 The MUC1-C homolog has also been linked to SRC activation and thereby subunit spans the cell membrane and includes a 58-amino-acid trastuzumab resistance in breast cancer cells and in breast (aa) extracellular domain, a 28-aa transmembrane domain and a tumors.14 Additional mechanisms of resistance have included 72-aa cytoplasmic domain (CD). MUC1-C associates in part with expression of a truncated p95HER2 that lacks the trastuzumab- RTKs through extracellular galectin-3 bridges.26 In addition, the binding domain,15 heterodimerization with other RTKs16–18 and MUC1-C-CD functions as a substrate for phosphorylation by RTKs downregulation of HER2 expression.19 Other studies have shown and SRC.24,25 The MUC1-C-CD also contains a YHPM motif that, 1Genus Oncology, Boston, MA, USA; 2Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and 3Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Correspondence: Dr D Kufe, Medical Oncology, Dana-Farber Cancer Institute, Harvard, Medical School, 450 Brookline Avenue, Dana 830, Boston, MA 02215, USA. E-mail: [email protected] Received 10 April 2013; revised 24 June 2013; accepted 25 June 2013; published online 5 August 2013 MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3423 when phosphorylated on tyrosine, functions as a binding site for performed on SKBR3 and BT474 breast cancer cells that phosphatidylinositol-3 kinase p85 SH2 domains.27 These findings overexpress HER2 and are dependent on phosphorylated HER2 and the demonstration that MUC1-C overexpression is sufficient to (p-HER2) for growth and survival.3 Levels of HER2 and MUC1-C induce anchorage-independent growth and tumorigenicity28,29 were found to be similar in these cells (Figure 1a). Co- provided the basis for developing agents that block the MUC1-C- immunoprecipitation studies further demonstrated that MUC1-C transforming function.25 In this way, the MUC1-C-CD contains a associates with HER2 in both SKBR3 and BT474 cells (Figure 1b). To CQC motif that is necessary for its homodimerization and assess the potential effects of MUC1-C on HER2 signaling, we function.30 Notably, cell-penetrating peptides that bind to the stably silenced MUC1-C in SKBR3 cells (Figure 1c, left). Notably, MUC1-C CQC motif are effective in inhibiting growth and inducing MUC1-C silencing was associated with the downregulation of death of human breast cancer cells growing in vitro and as p-HER2, but not HER2, abundance, consistent with a decrease in xenografts in mice.31 However, the effects of MUC1-C inhibition on HER2 activation (Figure 1c, left). In concert with these results, (i) the interaction between MUC1-C and HER2, and (ii) HER2 silencing MUC1-C in BT474 cells similarly resulted in suppression signaling in breast cancer cells are not known. of p-HER2 levels (Figure 1c, right). MUC1-C silencing was also The present studies demonstrate that MUC1-C contributes to associated with a decrease in SKBR3 cell growth (Figure 1d, left) HER2 activation in HER2-overexpressing breast cancer cells and and BT474 cell growth (Figure 1d, right). Moreover, colony thereby promotes their growth and clonogenic survival. The formation was substantially decreased by silencing MUC1-C results also demonstrate that the formation of MUC1-C/HER2 expression in both SKBR3 (Figure 1e, left) and BT474 (Figure 1e, complexes is substantially increased in the setting of trastuzumab right) cells. These findings indicated that MUC1-C contributes to resistance and that targeting MUC1-C in trastuzumab-resistant HER2 activation and proliferation of HER2-overexpressing breast cells results in the downregulation of HER2 activation. In concert cancer cells. with these findings, we show that targeting MUC1-C is effective in reversing trastuzumab resistance. The MUC1-C inhibitor, GO-203, downregulates HER2 phosphorylation The 72-aa MUC1-CD contains a CQC motif that is necessary for its RESULTS homodimerization30,32 (Figure 2a). The results of pull-down studies Silencing MUC1-C suppresses HER2 activation using lysates from SKBR3 cells demonstrated that MUC1-CD is MUC1 associates with HER2 in non-HER2-amplified breast cancer sufficient for forming complexes with HER2 (Figure 2a, left). cells and this interaction is increased by heregulin stimulation.23 However, binding to HER2 was not detectable with MUC1-CD in However, the functional significance of the MUC1-C/HER2 which the CQC motif had been mutated to AQA (Figure 2a, left). interaction has remained unclear. Accordingly, studies were Similar results were obtained when pull-down experiments were Figure 1. Silencing MUC1-C downregulates HER2 activation and colony formation. (a) Lysates from SKBR3 and BT474 cells were immunoblotted (IB) with the indicated antibodies. (b) Lysates from SKBR3 (left) and BT474 (right) cells were precipitated with anti-MUC1-C or a control immunoglobulin G (IgG). The precipitates were immunoblotted with the indicated antibodies. (c). SKBR3 (left) and BT474 (right) cells were infected with lentiviruses to stably express a control shRNA (CshRNA) or a MUC1shRNA. Lysates were immunoblotted with the indicated antibodies.
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